Rachael Hardison, PhD, shares recently published work from her graduate studies in the Mason and Justice Labs in The Research Institute at Nationwide Children’s.

Middle ear infections, also known as otitis media (OM), remain a health care concern for children in the United States and across the world, despite recent therapeutic and technological advances. For the subset of children with chronic or recurring infections, OM becomes a significant socioeconomic burden for caregivers. Chronic or recurrent episodes of OM can have unwanted complications, including hearing and developmental delays, the need for surgical intervention, and additional antibiotic prescriptions.

Nontypeable Haemophilus influenzae, or NTHI, is a gram-negative bacterium that is a major cause of these recurrent episodes of OM. In many cases, NTHI can persist and continue to cause disease despite repeated antibacterial therapies, and, alarmingly, the same NTHI strain can show up again even after periods of culture-negative results from the patient.

Scientists still don’t have a full picture of what makes NTHI so persistent in certain cases. So how can we design better therapeutics to directly target these persistent infections? The answer may lie in understanding how NTHI adapts to periods of nutrient starvation in the environment of the upper respiratory tract during OM.

NTHI requires the essential nutrient heme-iron for growth, but it can’t synthesize heme itself. As a result, NTHI must take up and metabolize available heme-iron in the human body in order to survive and grow. This isn’t as easy as it might sound, because the human body has a variety of ways to limit microbial access to heme-iron and other transition metals in sterile areas (such as the middle ear) by a process known as nutritional immunity.

By using short periods of heme-iron restriction when culturing NTHI, we can model nutritional immunity in a lab setting to investigate how adaptation to nutrient restriction affects NTHI pathogenesis. Our research team, co-headed by Kevin Mason, PhD, and Sheryl Justice, PhD, principal investigators in the Center for Microbial Pathogenesis, recently published two studies demonstrating a role for bacterial adaptation to nutrient limitation in inducing persistence and survival of NTHI as an intracellular pathogen inside middle ear epithelial cells.

It’s an exciting and unexpected finding that NTHI can adapt to heme-iron starvation and then survive inside the cells of the middle ear in what are known as intracellular bacterial communities (IBCs). This is especially so because NTHI is not classically considered to be a professional intracellular pathogen.

The study recently published in mSphere found that culturing NTHI under conditions of transient heme-iron restriction promotes invasion of NTHI into epithelial cells and subsequent escape from endocytic pathways that would typically result in bacterial killing in the cell. Specifically, NTHI that are taken up into the cell by macropinocytosis, a form of non-specific endocytosis, escape directly to the cell cytoplasm and form IBCs.

IBCs have been shown to be associated with recurrence and persistence of uropathogenic Escherichia coli (UPEC) and are fundamental to UPEC pathogenesis during urinary tract infection. Using UPEC as a paradigm, it’s tempting to speculate that life inside the epithelial cells may protect NTHI from the body’s immune defenses and/or antibacterial therapies and could ultimately promote reinfection or recurrence of OM. An exciting result of this study was that use of a drug that blocked macropinocytosis in the cells was sufficient to prevent NTHI from forming IBCs during infection. The therapeutic potential of this finding was recently highlighted in Science Translational Medicine.

However, we still needed to understand what benefit the IBC confers to NTHI and how it drives persistence and recurring infection. And we needed to identify bacterial factors that allow NTHI to quickly and efficiently adapt to changes, such as heme-restriction, in the microenvironment that promote survival inside the cell.

To that end, the second, related study recently published in PLoS Pathogens described the identification of a mutation that NTHI acquires after transient heme restriction. This mutation, in a gene called icc, is correlated with increased IBC formation in a preclinical model of otitis media.

Our studies link a mutation in icc with significantly increased long-term survival of NTHI in culture as well as with a higher IBC count during infection in the preclinical model. Icc encodes a cyclic AMP phosphodiesterase that functions to regulate the expression of a number of downstream genes, including those involved in DNA uptake, metabolism and virulence in several different pathogens. Following transient heme-iron restriction, NTHI acquired a mutation in icc in multiple independent experiments in the lab. Having the mutation increased the survival and persistence of the bacterial population compared to NTHI that did not harbor the mutation. We also sequenced clinical isolates from patients with OM and found mutations in icc in 10 percent of the clinical samples, suggesting a biological relevance for this mutation in NTHI-mediated infections.

The results of these studies begin to provide novel insight as to why some kids might struggle with recurring ear infections, and reveal a new avenue for investigating ways to eradicate intracellular NTHI. We are currently continuing investigations to further elucidate the benefits of IBCs and identify the factors that allow NTHI to “hide” inside epithelial cells. Ultimately, we’re hopeful that this will increase our understanding of how OM therapeutics could be improved or redesigned to prevent recurrent OM and its resulting complications.